Unlike other renewable energy sources, biomass can be converted directly into liquid fuels. The two most common types of biofuels are ethanol (ethyl alcohol) and biodiesel. Ethanol is an alcohol, which can be produced by fermenting any biomass high in carbohydrates (starches, sugars, or celluloses) through a process similar to brewing beer, once fermentable sugars have been obtained from the biomass material. The breakdown of the biomass into monomers (monosaccharides) requires the material to be softened through pretreatment, enzymes be added that hydrolyze the polymeric forms of sugars contained in the biomass into monosaccharides, and fermentation of both the 6-carbon and 5-carbon sugars to ethanol or to other desired bio-products.
Ethanol production in the United States grew from just a few million gallons in the mid-1970s to over 3.9 billion gallons in 2005. National energy security concerns, new Federal gasoline standards, and Government incentives have been the primary stimuli for this growth. Ethanol is mostly used as a fuel additive to cut down a vehicle's carbon monoxide and other smog-causing emissions. Flexible-fuel vehicles, which run on mixtures of gasoline and up to 85% ethanol, are now available.
Ethanol production received a major boost with the passage of the Clean Air Act (CAA) Amendments of 1990. Provisions of the CAA established the Oxygenated Fuels Program and the Reformulated Gasoline Program in an attempt to control carbon monoxide and ground-level ozone problems. The desires to improve air quality and enhance energy security have encouraged an increased demand for ethanol.
The majority of ethanol produced in the U.S. is produced from starch obtained from maize (maize) grain. It is anticipated that current starch supplies will be insufficient to meet future demands for fermentable sugars. Ligno-cellulosic biomass, such as stover, can be used as an alternative source of fermentable sugars for the production of ethanol.
Stover consists of all parts (leaves, stalks; with the exception of kernels) of plants such as maize, sorghum, soybean, sugarcane, or other plants that are left in the field after the harvest. This stover biomass is rich in cellulose and hemicellulose, which are cell-wall polysaccharides that can release fermentable sugars upon treatment with enzymes (such as cellulases). The biomass conversion of maize stover is currently not very cost-effective.
One strategy to improve the overall efficiency of converting stover biomass to ethanol is to modify the chemical composition of maize stover, specifically the plant cell wall polymer lignin. Lignin has been shown to shield the cellulose, resulting in reduced access by cellulases. In addition, lignin has been shown to be an inhibitor of cellulases.
Brown midrib (bm) mutants of maize, easily identified by the reddish-brown color of their central leaf vein, have been known for more than 75 years. Similar mutants have been found in sorghum, sudangrass, and pearl millet (Barriere and Argillier, 1993, Agronomie 13: 865-876). These mutants have generated significant agronomic interest because their tissues are more easily digested by ruminants, providing better nutrition for livestock (Cherney et al., 1991, Adv. Agron. 46: 157-198). However, the known varieties of these plants are not widely grown as they often suffer from slow growth, increased susceptibility to pests, and an increased tendency to lodge, all of which lead to decreased yields.
The maize brown midrib mutations, specifically the bm1 mutation, result in the production of abnormal lignin (Kuc and Nelson, 1964, Arch. Biochem. Biophys. 105: 103-113). Four independent maize bm mutants are known, each affecting the lignin biosynthetic pathway. Bm1 mutants have reduced expression of cinnamyl alcohol dehydrogenase (CAD) activity (Halpin et al., 1998, Plant J. 14: 545-553). The CAD enzyme converts cinnamyl aldehydes to their alcohol derivatives in the last step of monolignol synthesis. The reduction of CAD activity in maize bm1 mutants leads to large increases in the hydroxycinnamylaldehyde content of the lignin (Provan et al., 1997, J. Sci. Food Agric. 73: 133-142), and such aldehydes have been implicated in formation of the red chromophore responsible, in part, for the defining coloration of the mutants (Higuchi et al., 1994, J. Biotechnol. 37: 151-158).
Two independent mutations have been identified in the O-methyltransferase (OMT) gene of bm3 maize (Vignols et al., 1995, Plant Cell 7: 407-416). The bm3 gene encodes caffeic acid O-methyltransferase, or more accurately named, a 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferase (Humphreys et al., 1999, Proc Natl Acad Sci USA 96: 10045-10050). This enzyme catalyzes the methylation of the 5-position hydroxyl group of 5-hydroxyconiferyl aldehyde and 5-hydroxyconiferyl alcohol in monolignol synthesis. The net result of the bm3 mutation is a reduction in both the total amount of lignin deposited in the cell wall and a shift in lignin composition away from syringyl-type lignin because conversion of 5-hydroxyconiferyl aldehyde and 5-hydroxyconiferyl alcohol to sinapyl aldehyde and sinapyl alcohol, respectively is required for syringyl lignin synthesis. In bm3 mutants, the lignin also contains increased amounts of 5-hydroxyguaiacyl units, but in addition has a greatly reduced ratio of syringyl to guaiacyl units, as well as decreases in p-coumaric acid esters and overall lignin content (Chabbert et al., 1994, J. Sci. Food. Agric. 64: 349-355).
To improve the efficiency of converting biomass to ethanol, it would be advantageous to increase the amount of fermentable sugars in a plant stover. This invention provides that and related needs.